In the field of peptide synthesis certain couplings are known as difficult couplings, especially those involving coupling to bulky or sterically hindered amino acid residues, such as N alkylated, C-alkylated and Cα branched amino acids. In order to obtain acceptable yields when these couplings are performed a variety of special coupling reagents have been developed. Among other known procedures, is the use of pre-formed amino acid chlorides to improve the outcome of the coupling reactions.
The general use of protected amino acid chlorides in solid phase peptide synthesis (SPPS) is limited mainly because of the fact that chlorides of fluorenylmethoxycarbonyl (Fmoc) amino acid having side chains protected with acid labile protecting groups, including but not limited to t-butyl (t-Bu), t-butoxycarbonyl (Boc) or trityl (Trt), have limited shelf stability. For example, chlorides of Fmoc-amino acids (AAs) with t-Bu-protected side chains could not generally be accommodated. In some cases (aspartic acid and glutamic acid) the chlorides could not be obtained and in other cases (tyrosine, serine, threonine) their shelf stability appeared insufficient for practical utilization. In addition, the preparation of chlorides derived from Fmoc-Lysine(Boc), Fmoc-Tryptophan (Boc), Fmoc-Cysteine(Trt), Fmoc-Glutamine(Trt) and Fmoc-Arginine 2,2,5,7,8-Pentamethyl chroman-6-sulphonyl (Pmc) is problematic because of side reactions and require special reaction conditions and purification (Carpino et al. Acc. Chem. Res. 29:268, 1996). This problem also hampers the general use of pre-formed Fmoc amino acid chlorides in automatic peptide synthesis. Despite these limitations, acid chlorides were used in SPPS especially for the assembly of hindered secondary amino acids (see Carpino et al. 1996 ibid and refs. within).
Coupling of protected amino acids to Nα-alkylated amino acids was previously considered to be a difficult coupling both in solution and solid phase. This coupling was used in model peptides to demonstrate the efficiency of new, more effective, coupling methods. In these models, N-Methylated amino acids were used as nucleophiles, since coupling to N-Methylated amino acids having steric hindrance on the Cα (e.g., N-methyl valine and N-methyl Aminoisobutyric acid) was found to be much slower than to proline. Certain coupling agents and activation methods such as bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP) (Coste et al. Tetrahetron Lett. 31 669, 1990), 1-hydroxy-7-azabenzotriazole (HOAt)/O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (Carpino et al. J. Chem. Soc., Chem. Commun. 201, 1994), urethane-protected N-carboxyanhydrides (UNCA) (Spencer et al. Int. J. Pep. Prot. Res. 40:282, 1992) and acid halides (Carpino et al., 1996 ibid) were specially recommended to achieve coupling to N-alkyl amino acids.
The acid chloride method was found to be a superior way to couple protected amino acids to sterically hindered amino acid derivatives, such as the N-alkyl amino acids during SPPS of backbone cyclic peptides. To overcome the limitations of the pre-formed acid chloride method and to allow its general use in SPPS, it would be advantageous to have an efficient and generally applicable method allowing the in-situ generation of Fmoc-AAs chlorides.
The reagent bis-(trichloromethyl)carbonate (BTC) (Councler, C. Ber. Dtsch. Chem. Ges. 13:1697, 1880) also named hexachlorodimethyl carbonate or “triphosgene” is a solid stable phosgene substitute equivalent to three moles of phosgene. Triphosgene has been used as an efficient carbonylating agent for liquid and solid phase synthesis of various aza-analogues of peptides containing aza-alanine, aza-aspartic acid and aza-asparagine residues (Andre et al. J. Pep. Sci. 3:429, 1997).
The use of triphosgene as a reagent for formation of isocyanates or other reactive species useful in peptide chemistry has also been disclosed (Eckert DE3440141, Nippon Kayaku JP10007623). The usefulness of triphosgene in preparation of various intermediates for pharmaceuticals has also been disclosed (Hoffmann et al. DD292452).
It is neither taught nor suggested in the art that the triphosgene reagent is suitable for the in-situ generation of protected amino acid chlorides, namely as a coupling agent in SPPS (for review see Cotarca et al. Synthesis 553, 1996).
Phosgene gas has long been a valuable asset to both lab and plant scale operations however the dangers of using it are also well documented, especially the respiratory hazards. Liquid trichloromethyl chloroformate, commonly known as “diphosgene” (Fridgen, L. N. and Prol, J. J., J. Org. Chem. 54:3231, 1989) which has already been used as a phosgene substitute, has proven useful in all common phosgene reactions, but being a liquid its transport and storage still impose considerable hazard. Being a crystalline solid (mp 81–83° C.), BTC is safer and easy to handle and therefore became the reagent of choice for all applications where phosgene chemistry is required (Cotarca et al. ibid). Synthetically, one mole of BTC yields three mole-equivalents of phosgene which reacts with hydroxyl, amine or carboxylic acid nucleophiles forming chloroformate, isocyanate or acyl chloride, respectively.
Considering all these features together with the fact that BTC is inexpensive and less susceptible to hydrolysis than phosgene, it is surprising that the use of BTC as a general coupling agent has not been considered.
Backbone Cyclized Peptide Analogs
Backbone cyclization is a concept that allows the conversion of peptides into conformationally constrained peptidomimetics with desired pharmacological properties such as metabolic stability, selectivity and improved bioavailability (Gilon et al. Biopolymers 31:745, 1991; Byk et al. J. Med. Chem. 39:3174, 1996; Gilon et al. J. Med. Chem. 41:919, 1998).
In backbone cyclization the Nα and/or Cα atoms in the peptide backbone are linked through various spacers. To synthesize N-backbone cyclic peptides a large number of orthogonally protected-functionalized Nα alkyl amino acids (N-building units) were prepared (Bitan et al. J. Chem. Soc., Perkin trans. I 1501, 1997a; Muller et al. J. Org. Chem. 62:411, 1997). These units were incorporated into peptides by SPPS or solution methodologies and after orthogonal removal of the protecting groups from the ω-functions on the Nα-alkyl they are cyclized. A critical step in the synthesis of N-backbone cyclic peptides is the coupling of protected amino acids to the sterically hindered secondary amine of the Nα (ω-functionalized alkyl) amino acid residue on the peptidyl-resin.
The synthesis of N-backbone cyclic peptides that incorporate Nα (ω-functionalized alkyl) Glycine building units were reported previously. In these cases couplings of the protected amino acids to the secondary amine of the Gly building unit attached to peptidyl resin were achieved by multiple couplings with benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexaflourophosphate BOP or PyBroP as coupling agents (Bitan et al. J. Pept. Res. 49:421, 1997b; Byk et al., 1996 ibid).
Generally, the coupling of protected AAs to building units other than Gly (non-Gly backbone cyclic building units) were found to be difficult and even impossible.
It has been shown that the coupling of many Fmoc AAs to sterically hindered secondary amines including a variety of non-Gly building units attached to peptidyl-resin could be achieved in moderate to high yields using the acid-chloride method but not acid fluorides (Carpino et al. 1996 ibid) or other coupling agents such as PyBrOP (Coste et al., ibid), HOAt/HATU Carpino 1994 ibid), 2-(2-Oxo-1(2H)-pyridyl)-1,1,3,3-bispenta-methyleneuronium tetrafluoroborate (TOPPipU) (Henkleinet al. In ““Peptides 1990” Proc. of the 21th European Peptide Symposium”, E. Giralt and D. Andreu, eds, pp. 67. ESCOM Leiden, 1990), UNCA (Spencer et al., 1992) and Mukaiyama reagent (Mukaiyama, T. Angew. Chem., Int. Ed. Ingl. 18:7078, 1979).